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Ovarian steroid : effects on immune responses and Chlamydia trachomatis infections of the female genital tract

LM Hafner1, K Cunningham1 and KW Beagley1

Female sex hormones are known to regulate the adaptive and innate immune functions of the female reproductive tract. This review aims to update our current knowledge of the effects of the sex hormones estradiol and in the female reproductive tract on innate immunity, antigen presentation, specific immune responses, antibody secretion, genital tract infections caused by Chlamydia trachomatis, and vaccine-induced immunity.

INTRODUCTION depicts the FGT anatomy and the location and relative A critical function of the unique mucosal immune system of the abundances of innate immune cells at this mucosal site.6,8–15 female genital tract (FGT) is to identify and eliminate In the FGT, the major lymphocyte components are natural potentially pathogenic viral and bacterial agents and to provide killer (NK) cells and T lymphocytes, including cluster of protection against sexually transmitted diseases. Globally, it has differentiation (CD) 3 þ T lymphocytes that are present in all been estimated that in adults between 15 and 49 years of age the tissues of the tract. In the LGT, the CD8 þ and CD4 þ are there were 105.7 million cases of new Chlamydia trachomatis dispersed throughout the stroma while lymphoid aggregates of sexually transmitted infections (STIs) in 2008.1 Significant these cells are formed in the .16 Granulocytes are disease sequelae following chlamydial infections of the FGT present and these are principally located in the fallopian include pelvic inflammatory disease, tubal infertility, and tubes. Finally, relative to T lymphocytes, smaller numbers of ectopic .2–4 monocytes and B lymphocytes are found throughout all tissues The FGT comprises several immune compartments found in of the FGT.17 the upper genital tract (UGT; endocervix, , fallopian Immune cells at this site express both surface and cytosolic tubes, and uterus) and lower genital tract (LGT) (ectocervix and pattern recognition receptors (PRRs), including among ). The UGT with Type I mucosa5 is lined with a single others Toll-like receptors (TLRs) and the nucleotide-binding layer of columnar and the LGT with Type II mucosa oligomerization domain (NOD)-like receptors. TLRs 2 and 4 is lined with stratified squamous epithelium (reviewed in are highly expressed in the UGT (fallopian tubes and Pudney et al.6). The transitional zone where squamous ) when compared with expression levels in the LGT epithelium changes to columnar epithelium is the most ( and ectocervix) while TLRs 7, 8 and 9 and immunologically active site of the FGT. Although currently NOD-1 (which is involved in sensing Chlamydia infection) and documented (relative) microbial sterility is found in the NOD-2 are expressed at similar levels throughout the tract.18–20 uterus and fallopian tubes, commensal such as In addition to providing a physical barrier for protection, Lactobacillus crispatus are found in the LGT and are under the genital tract epithelial cells (ECs) lining the FGT are hormonal influences at this site.7 immunologically active cells. For example, they act as key The human FGT contains components of the innate and initiators of innate and adaptive immunity by secreting adaptive mucosal immune responses that are found in various cytokines and chemokines, including interleukin (IL)-1b, distributions at different sites throughout the tract. Figure 1 tumor necrosis factor a (TNF-a), IL-6, IL-8, and GM-CSF

1Infectious Diseases Program, Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), Kelvin Grove, Queensland, Australia. Correspondence: LM Hafner ([email protected]) Received 5 June 2011; accepted 4 June 2013; published online 17 July 2013. doi:10.1038/mi.2013.46

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Figure 1 The female genital tract: anatomy and innate immune cells of the genital mucosa. This figure depicts non-immune and innate immune cells found throughout the female genital tract, including macrophages, dendritic cells, neutrophils and natural killer (NK) cells. Immune cell receptors are shown on immune cells. Note that epithelial cell receptors CD4, C-C motif chemokine receptor 5 (CCR5), C-X-C motif chemokine receptor 4 (CXCR4) and GalC on endometrial and endocervical cells are not shown in this figure. TLR, Toll-like receptor; NKG2D, an activating receptor expressed on NK cells that binds to MHC class 1 chain–related protein A and B (MICA and MICB).

(granulocyte-macrophages colony-stimulating factor), in The recruitment and function of immune components of the response to human chlamydial infections.21 FGT are precisely regulated by hormonal changes during the Host protection of the FGT is afforded by two arms of menstrual cycle by the sex steroids that co-ordinate cell defense comprising innate and acquired immunity. The cellular trafficking and immune activation at this mucosal site. Female components of specific immune responses at this site are T sex hormones , progestins, and androgens are lymphocytes expressing ab- and gd-T-cell receptors (TCR) and produced by ovarian cells with levels of estradiol (E2 or immunoglobulins (Igs) producing B lymphocytes.22 Effectors 17b-estradiol) and progesterone or P4 (pregn-4-ene3, of non-specific responses include ECs, monocytes, macro- 20-dione) varying in accordance with fluctuations in the phages, granulocytes (neutrophils, eosinophils, and basophils), menstrual cycle.22 In the menstrual/ovarian cycle of humans, dendritic cells (DCs), and NK cells that confer protection due to the endometrium develops and follicles grow until ovulation in chemokines, cytokines, and phagocytosis. Cells of both the the Proliferative/Follicular phase. The Secretory/Luteal phase is innate and adaptive immune systems are found in fallopian characterized by high levels of progesterone from the corpus tubes, uterus, cervix, and vagina.23 Localization of T-lymphocyte luteum to maintain the endometrium. Finally, populations in the female UGT and LGT over the menstrual regression and menstruation occurs during the menstrual cycle is summarized in Table 1.10–15,24–40 phase. At menstruation, serum E2 levels measure typically Innate host defense, particularly in the LGT, is provided by o50 pg ml À 1. Gradually, increasing amounts of E2 are found the vaginal microbiota maintained in a healthy equilibrium in the follicular (proliferative) phase of the adult female (reviewed in Farage et al.41) as well as by the myriad of menstrual cycle with serum levels at a pre-ovulatory stage antimicrobial peptides and protease inhibitors of cervicovaginal (day 14) ranging from 110–410 pg ml À 1 and dropping briefly fluid (Cole42 and reviewed in Farage et al.41 and Wira et al.43). at ovulation. Levels of E2 (20–160 pg mol À 1) and P4 The antimicrobial peptides and PRR TLRs are key inter- (45ngmlÀ 1) are present during the luteal (secretory) phase mediaries of innate immunity in the FGT.43–46 from days 14–28 of the menstrual cycle typically peaking at day

860 VOLUME 6 NUMBER 5 | SEPTEMBER 2013 | www.nature.com/mi Mucosal Table 1 Localization of T lymphocytes in the female upper (UGT) and lower (LGT) genital tracts: effects of menstrual cycle stage and chlamydial infection

Immunology Menstrual cycle stage Upper genital tract (UGT) Lower genital tract (LGT) (ovarian phase) Fallopian Proliferative Secretory Chlamydia trachomatis T lymphocytes Ovaries tubes Uterus/endometrium Endocervix Ectocervix Vagina (follicular) (luteal) genital tract infection References | OUE6NME 5 NUMBER 6 VOLUME Serum estradiol 220 pmol l À 1 o130 pmol l À 1 Progesterone o2ngmlÀ 1 45ngmlÀ 1

T helper cells CD45 þ ,CD3þ , CD45 þ cells CCR5 þ CD4 þ CD3 þ , CD3 þ CD4 þ (30–40% CCR5 þ ( þ ) CCR5 þ ( þ ) Endocervix: CD4 þ cells Kaldensjo¨ 24 CD4 þ Th1, Th2, CD3 þ CD8 þ CD3 þ CD45 þ CD4 þ CD4 þ of vaginal T cells) CD45 þ leu- CD45 þ leu- ( þþþþ) Hamad25 | ETME 2013 SEPTEMBER Th3, Th17 or TFH 4% than CD3 þ kocytes ( þ ) kocytes ( þ ) Endometrium CCR5 and Shaw et al.26 CD3 þ CD4 þ (mid-luteal) (mid-luteal) CD103 cells ( þ ) Cervix Dispep et al.27 CD3 þ CD4 þ Ficarra28 Johansson et al.29 Kamat & Isaacson30 Starkey et al.31 Vassiliadou & Bulmer32 Agrawal et al.35

CytotoxicT cells CD45 þ ,CD3þ , þ CD3 þ CTL þþ þm (uterus) k(uterus) White et al.33 CD8 þ CD8 þ CTL CD8 þ CTL activity ( þþþ) 450% kCTL activity CD8 þ cells Cervix White et al.33 activity vaginal T cells (no change in CD3 þ CD8 þ Li et al.34 CD8 þ cell Ficarra28 number) Johansson et al.29 Agrawal et al.35

CD8 þ CTL CTL activity CTL activity Measurable Measurable White et al.36

CD8 þ CD4 À Yeaman et al.16

Treg cells Natural CD4 þ , þ mLevels kLevels Increased expression of Arrivito et al.37 CD25 þ FoxP3 þ correlating significant FoxP3 þ cells primarily Tai et al.38 Adaptive Tr1 and Th3 with E2 after ovulation in UGT Marks et al.39 k

(uterus) Levels (E2 at REVIEW mLevels (E2 at physiological physiological doses) doses) 861 REVIEW

21 of the cycle. At the end of the secretory phase, E2 levels return 40

¨ to their menstrual levels. et al.

24 The sex hormones E2 and P4 are key contributors to susceptibility and innate and acquired immune responses to Kaldensjo et al. Kelly bacterial and viral infections of the FGT47 (reviewed in Bouman et al.,22 Rakasz et al.48 and Butts et al.49). It is also known that

þ these hormones contribute to the sex-based differences both in 7

b innate and in acquired immunity and infectious diseases with þ 4 a estrogens affecting levels of chemokine receptors by T cells and the higher CD4 þ T-cell populations in women undergoing significant changes during the ovarian cycle (reviewed in 50 genital tract infection References

Chlamydia trachomatis Fish ). Differences in E2 levels between male and female mice Endocervix: cutaneous lymphocyte antigen (CLA) have also been reported as impacting on the expression of inducible nitric oxide synthase (iNOS)51 and hence the produc- tion of nitric oxide (NO), a gas that modulates production of pro-inflammatory cytokines, such as IL-6 and TNF-a.52

Secretory (luteal) Innate and adaptive mucosal immunity in the FGT are regulated by the female sex hormones E2 and P4 and have been reviewed in Bouman et al.22 and Butts and Sternberg.53 (ovarian phase) Hormones regulate the transport of Igs, the levels of cytokines, Menstrual cycle stage 54,55 (follicular) the expression of TLR genes, and the distribution of Proliferative immune cells and antigen presentation in the genital tissues during the reproductive cycle23,44 Figure 2 depicts the menstrual cycle phases, sex steroid levels, and

) symbols indicating increased cell numbers. immune cells present in the FGT. þ The afferent and efferent arms of uterine, cervical, and vaginal immune responses are separately regulated and the control by sex hormones is distinct and species-specific in vaginal immune compartments.48 In humans, uterus/cervix cellularity and CTL activity is menstrual cycle–dependent with abundant IgA-secreting cells and secretory component expres- sion in the endocervix. In the vaginal immune compartment, cellularity and CTL activity is not menstrual cycle–dependent, CD8 þ T cells dominate, (and these are vital in a response to chlamydial infection),48 there is only a minor population of þ IgA plasma cells and low levels of secretory component expression, and IgG dominates with peri-ovulatory antibody þ peaks.48

CD4 This review summarizes our current knowledge of the effects of sex hormones E2 and P4 in the FGT on innate and adaptive

IELs stromal CCR5 immunity, antibody secretion, genital tract infections caused by C. trachomatis, and on vaccine-induced immunity.

HORMONAL EFFECTS ON INNATE IMMUNITY tubes Uterus/endometrium Endocervix Ectocervix Vagina

Fallopian There are many components of innate immunity operational Upper genital tract (UGT) Lower genital tract (LGT) in the vagina and these have recently been reviewed45,56 as have components of innate immunity in the and endometrium.57 These include soluble factors (such as mannose-binding lectin, complement components, defensins, secretory leukocyte protease inhibitor (SLPI), and nitric oxide), membrane-associated components such as Toll-like- dependent and -independent receptors, as well as phagocytic cells. The innate immune system of the FGT is also comprised of many cells, including macrophages, DCs, neutrophils, NK

T cells cells, and ECs. It is now becoming increasingly apparent that all ) symbol indicates the presence of T cells in the UGT and LGT over the menstrual cycle with increasing numbers of ( Intraepithelial lymphocytes (IELs) Memory T cells þ gd Table 1 Continued CCR, C-C motif chemokine( receptor; CD, cluster of differentiation; CTL, cytotoxic T lymphocyte; TFH, T follicular helper; Th, T helper. T lymphocytes Ovaries aspects of innate immunity, including secretion of protective

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Figure 2 Menstrual cycle phases, sex steroid levels and effects on innate immune cells in the female genital tract. This figure depicts the immune cell types and their relative abundances across the E2 dominant (proliferative) and P4 dominant (secretory) phases of the menstrual cycle.

Table 2 Innate immunity in the human FGT

Lower genital tract (LGT) (ectocervix and vagina) Proliferative (E2 dominant) Secretory (P4 dominant) References Soluble factors (AMPs, HBD2, elafin) kHBD2 and elafin mSLPI 7,23 Epithelial cells (multilayered, squamous) Thick Thinner vaginal epithelium 16 Neutrophils ( ¼ )(¼ ) 53,59 Macrophages Vagina ( ¼ ) Vagina ( ¼ ) 59 NK cells (CD45 þ , CD56 þ CD3 À CD16 þ )(¼ )(¼ ) 26,31

Upper genital tract (UGT) (Endocervix and Proliferative (E2 dominant) Secretory (P4 dominant) References endometrium, fallopian tubes, and ovaries) Soluble factors (AMPs, HBDs, elafin) mHBD4 (endometrium) mSLPI, HBD1, HBD3 60–62 (note: HBD2 and elafin peak (endometrium) (maximal during menstruation) SLPI expression) and elafin (fallopian tubes— constitutively expressed) PRRs (TLR, NOD-1, 2) TLR10 (fallopian tubes) TLRs TLRs 2, 3 5, 6, 9, including 55,63–68 1–7 but not TLR4 TLR4)m(endometrium), (endocervix) TLR10m(fallopian tubes) Epithelial cells (single layer, columnar) Thick . Thin uterine epithelium 16,69 Constitutively express IFN-e (Type 1 IFN) in luminal and glandular cells highest in the proliferative phase Neutrophils ( ¼ ) Endometrialm 54,59,70 Macrophages Endometrialm (before 31 menstruation) Dendritic cells CD83 þ ( ¼ ) CD1a þ m 71 NK cells CD45 þ , CD56 þ CD3 À CD16 À , CD69 þ uNK mCTL activity Endometrial NK cells (with 10–15,26,31,40 CD94 þ NKG2D receptor) mMICA uNK kCTL activity via IL-18 AMP,antimicrobial peptide; CD, cluster of differentiation; CTL, cytotoxic T lymphocyte; FGT,female genital tract; HBD2, human b defensin 2; IFN, interferon; IL, interleukin; SLPI, secretory leukocyte protease inhibitor; NK, natural killer; NKG2D, an activating receptor expressed on NK cells that binds to MHC class 1 chain–related protein A and B (MICA and MICB); NOD, nucleotide-binding oligomerization domain; PRR, pattern recognition receptor; TLR, Toll-like receptor;uNK, uterine natural killer. The effects of menstrual cycle phase (proliferative or secretory) on the levels (m, increased; ( ¼ ), constant; or k, decreased) and functions (activity) of innate immune cells, non- immune cells, and soluble factors. factors and localization and function of innate immune and The innate response is triggered by PRRs (including TLRs non-immune cells, and receptor expression on ECs58 vary with and non-TLRs such as NOD-like receptors) expressed the release of ovarian steroid hormones over the menstrual predominantly by immune effector cells such as macrophages, cycle. Table 27,10–16,23,26,31,40,59–71 summarizes the effects of neutrophils, and DCs that are found in the genital mucosa. (E2) and Progesterone (P4) at the proliferative and These include TLR4 and its co-receptor CD14 (chlamydial secretory phases on the aspects of innate immunity in the FGT. lipopolysaccharide (LPS) and heat-shock protein 60

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(HSP60)),72–74 TLR2 (chlamydial HSP60),75,76 and NOD protein, P4 has been shown to inhibit many functions of DCs, e.g., Nod2 (rudimentary proteoglycan motif produced by C. tracho- suppressing TLR-mediated cytokine production by some matis ).77 Expression of TLR genes 2–6, 9, and 10 is reportedly subsets of DCs with a recent study focussing on the effect significantly higher in human endometrial tissue during the of this sex hormone on TLR signalling in DCs.89 These authors secretory phase (when P4 is the predominant hormone) compared state that P4 inhibition of plasmacytoid DC function may with all the other phases of the menstrual cycle.47,68 impair specific antiviral responses (e.g., HIV or Herpes simplex The epithelial layer of the vaginal epithelium (LGT) and virus (HSV)-2) that rely on certain TLR subsets and that this endometrium, endocervix, and fallopian tubes (UGT) is induced inhibition may have a significant impact on global health. P4 is to proliferate producing a thickened uterine epithelium during also a potent inducer of semi-mature tolerogenic DCs and is the proliferative stage of the menstrual cycle when increased reported to inhibit mature DC activity, which can ultimately secretion of the E2 hormone is observed. During the secretory contribute to the induction of pro-inflammatory responses.93 phase of the menstrual cycle when P4 levels exceed E2 levels, the The effect of sex steroids on the induction and function of a epithelial layer is reduced in thickness. The human FGT is not as recently described CD4 T-cell subset, Th17 T cells that are permeable as the rodent FGT to antigen uptake during the involved in chronic inflammation in some disease states,94 P4-dominated secretory phase as P4 does not significantly is also an area flagged for important future research, as reduce the thickness of the vaginal epithelium in humans.78 P4-mediated disease remission in autoimmune diseases may involve impaired DC-regulated induction of Th17 cells.89 The Rodent FGT inhibitory effects of P4 on immature bone marrow–derived In murine models, it has been reported that increased genital DCs (BMDCs) in female and male rodents has recently been infections with C. trachomatis are seen in progesterone-treated reported to differ and is likely due to differences in P4 receptor 88 mice.79 Thus the immune responses to chlamydial infections protein expression affecting DC function in these animals. seen in the FGT rodents and humans are likely to be modulated A recent review of the functions of DCs and subsets of in different ways by these vaginal permeability differences. macrophages in the FGT has also addressed the sex hormone regulation of these professional APCs at this mucosal site.95 Changes in the populations of DC and macrophage cells in the HORMONAL EFFECTS ON ANTIGEN PRESENTATION FGT occur due to (i) varying levels of ovarian steroid hormones In the FGT, sex hormones regulate both antigen presentation at this site,96 (ii) the delivery of CpG oligodeoxynucleotide to by DCs and also the numbers of macrophages and antigen- the murine genital tract,97 and (iii) the stages of the menstrual presenting cells (APCs) found within ECs of uterine and vaginal cycle, noting that the frequencies of macrophages and tissues.80,81 Human vaginal tissue contains cells that can immature DCs are higher in the menstrual phase when present antigen to memory T cells,82 and ECs from uterine compared with the other phases of the cycle.98,99 endometrium are known to constitutively express major In a study investigating genital tract infections of C57BL/6 histocompatibility complex Class II molecules that are required mice with C. trachomatis serovar D, it has been reported that in to present antigen to T lymphocytes.83 In the rat, E2 enhances the LGTs of these mice IL-10 (primarily from CD11b þ DC) antigen presentation by uterine ECs,80 whereas in the mouse E2 was the dominant IL, suggesting the presence of Th2 cells in inhibits EC antigen presentation.81 Mature DCs are character- these animals.39 In the UGTs of these same animals, a Th1 ized by high production of IL-1284 that facilitates the subset of cells dominated with increased levels of IFN-g development of gamma interferon producing Th1 cells.85 expression was recorded following genital tract infections of Estrogen has been shown to modulate the expression of these mice with C. trachomatis. This suggests that DCs are cytokines and chemokines in human monocyte-derived DCs, major contributors to IL-10 production in the LGT of mice and with mature DCs pre-treated with E2 showing an increased that production of this IL likely leads to an anti-inflammatory ability to stimulate naive CD4 þ T cells.86 E2 can therefore site of poor Th1 immunity at this mucosal site.39 regulate DC effector function and can induce and maintain CD16-expressing macrophages and monocytes are also key inflammatory responses in the FGT. components of the innate immune system of the FGT and are The effects of E2 on the differentiation and function of APCs cells that are capable of producing cytokines and chemokines in vitro has been reviewed (see Nalbandian and Kovats87 and upon activation. These cells are also known to be profoundly Butts and Sternberg88). Much of the current evidence shows affected by E2 (reviewed in Fish50). Studies have shown that E2 that E2 can activate DCs and differentially regulate adaptive uses both ERa and ER beta (ERb) to decrease CD16 expression immune responses through direct effects on DC functions (see on monocytes and hence to alter monocytic cytokine release Hughes and Clark89) via E2 receptor (ER) ligands (reviewed in following CD16 receptor activation.100 More recently, it has Kovats and Carreras90). It has been shown that ER-alpha (ERa) been shown that human macrophages predominantly express is the main receptor regulating E2-dependent DC differentia- an N-terminal truncated variant of ERa,ERa46, and that this is tion in murine bone marrow–derived DCs in vitro.91 At high also regulated by E2.101 Removal of endogenous E2 has been concentrations typical during pregnancy, E2 plus TNF-a have shown to reduce expression of TLR4 on the surface on murine been reported to induce a distorted maturation of human DCs macrophages thus decreasing both pro- and anti-inflammatory resulting in an increased capacity to initiate Th2 responses.92 cytokine releases from these cells. The addition of exogenous

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E2, however, corresponded with significantly higher expression the female UGT and LGT are T lymphocytes, specifically of cytokines from these cells highlighting the immuno- CD4 þ and CD8 þ T cells, along with APCs including enhancing effects of E2 on these cells.102 TLR4-mediated macrophages and DCs. Immune mediators of cell-mediated immune responses in macrophages have recently been reported immunity are found distributed within the distinct regions of to be inhibited by P4 via suppression of NF-kb activation, the female LGT. The cervix is a major inductive and effector site suggesting a mechanistic explanation for the regulation of for cell-mediated immunity in the lower human FGT. In innate immune responses of these cells by this sex hormone.103 women with no current FGT inflammation, macrophages and CD4 þ and CD8 þ T lymphocytes are most prevalent ROLES OF NON-CONVENTIONAL APCS IN UTERINE AND between the ecto- and endo-cervix, in the so-called cervical VAGINAL TISSUES IN INNATE IMMUNITY transformation zone, and also in the surrounding tissue. In the FGT, the underlying stromal cells are known to regulate Intraepithelial lymphocytes are predominantly CD8 þ T cells growth and function of polarized ECs,44 and uterine stromal in the cervical transformation zone. The normal vaginal cells mediate TNF-a release from uterine ECs in the presence of mucosa contains T cells and APCs with the few immune cells E2.104 It has also been reported that TLRs 1–10 expression levels present consisting primarily of CD1a þ DCs and CD8 þ in murine vaginal ECs were significantly increased during the intraepithelial lymphocytes.116 The tissues in the UGT have diestrous phase and expression of TLR2 was significantly been found to express a greater number Th1-associated decreased in ECs cultured in the presence of stromal cells.105 chemokines than the tissues in the LGT comprising the The role of ECs and their associated cytokines in innate cervical-vaginal region.116 The menstrual cycle and menopause immunity of the FGT has been well documented106 (reviewed have been reported to have no apparent effect on cellular in Quayle9). Sex hormones regulate the in rodents localization or abundance of T-lymphocyte subsets and APCs and the menstrual cycle in women to optimize mating and in any of the LGT tissues.6 fertility, and they are also involved in regulating innate Migration of T-lymphocyte subsets is vital for adaptive immunity in the FGT in both rodents and in mammals.104 immune responses and is the result of host–pathogen Human oviduct ECs express TLR 3, and E2 has been interactions. Effector lymphocytes must be recruited to the reported to suppress the TLR-3-induced cytokine and che- FGT after activation in the local iliac lymph nodes.6 To mokine production in human endometrial ECs.107 Chemo- eradicate pathogens from the infected genital tract, effector kines, including C-C motif chemokine ligand 20/macrophage T cells must migrate back to the FGT.6,116 Following infection inflammatory protein 3a (CCL20/MIP-3 a), have been found of the mucosal ECs of the FGT, the pathogen induces release of to have antimicrobial properties,108 and CCL20/MIP-3 a is host factors modifying the adhesion cascade. Following unique in binding exclusively to CCR6 (chemokine (C-C motif) chlamydial infection of the murine FGT, increased expression receptor 6) receptor in humans.109 Expression of CCL20/MIP-3 of an adhesion molecule117 and expression of Th-1-associated a has been reported from ECs of the FGT, and secretion chemokines118 has been reported. The release of host factors, in occurs constitutively as well as being induced by inflammatory turn, influences the recruitment of lymphocytes to the infection conditions.110 site. These host factors act to modify the specific T-lymphocyte E2 inhibits IL-1b-mediated responses by uterine ECs, subsets recruited to the FGT in response to infection. In suggesting that an inhibitory effect of E2 may be vital for humans, women with cervicitis and have been reducing the pro-inflammatory responses that are mediated by reported to have increased numbers of intraepithelial these cells.111 E2 also can act on endometrial ECs to suppress CD8 þ and CD4 þ lymphocytes and APCs in the vaginal TLR3-induced cytokines and chemokines.107 mucosae when compared with women having no symptomatic The effects of E2 and P4 on the release of antibacterial factors infections at these mucosal sites.116 by ECs in the rat uterus was studied, and it was reported that in In mice, investigations into the distribution and function of T ovariectomized rats treated with E2, antibacterial activity lymphocytes in the FGT have reported that 70–90% of the against Staphylococcus aureus and Escherichia coli increased lymphocytes in the uterus and upper parts of the cervix of in uterine secretions, an effect that was reversed with C57BL/6 mice are extrathymically derived CD3 þ abTCR þ P4 treatments.61 P4 has been reported to potentiate the CD4-CD8 À T cells.119 In the murine vaginal tract, 30–40% of E2-stimulated expression of NOS isoforms, including endothelial the T lymphocytes have been reported as being mostly NOS in endometrial-derived primary and human endometrial CD4 þ T cells (see mini-review by Hamad25). Hormonal surface EC line.112 P4 has also been reported to stimulate the control of cytokine secretion by vaginal and uterine lymphoid expression of NOS in endometrial ECs, and this may potentially cells has been investigated in two strains of mice (Balb/c affect infection outcomes at this mucosal site.113 and C57BL/6), and it was reported that the numbers of IFN-g-secreting vaginal and uterine lymphoid cells increased at HORMONAL EFFECTS ON T LYMPHOCYTES IN THE FEMALE diestrous in both strains of mice.120 Female C57BL/6 mice UGT AND LGT were also used to show that estrogen-conditioned bone The FGTs of humans and mice lack underlying organized marrow–derived DCs (BM-DC) could enhance regulatory lymphoid aggregrates,114 contrasting with reports of lymphoid T-cell (Treg) activity and also directly inhibit effector T cells in cell aggregates in the mucosa of the simian vagina.115 Present in the absence of Treg cells.121

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In humans, expansion of human CD4 þ CD25 þ Tregs has have a major part in making this mucosal site distinctively been reported to occur in the late follicular (proliferative) susceptible to chlamydial infections.127,128 phases of the menstrual cycles of fertile women.37 This increase The FGT of humans and of rodents differs in many ways that in Tregs was correlated not only with normal serum levels of E2 can modulate the immune responses at this mucosal site. Both in these women but also with an increased expression of the human and rodent genital tracts have large numbers of DCs; Foxp3 gene.38 Interestingly, E2 is reportedly responsible for the however, only the human FGT contains large numbers of elevated immune-suppressive potential of Tregs.122 CD4 þ , CD8 þ T cells, B cells, and macrophages. Leukocyte Increased expression of FoxP3 þ mRNA was seen primarily subpopulations are found throughout the human FGT and in the FGT of mice following infections of the genital tracts of include T helper cells (Th1, Th2, Th17), cytotoxic T cells, Treg these animals with C. trachomatis serovar D.39 It is generally cells, NK cells, and gd T cells. In contrast to rodents, the accepted that Treg cell expansion is modulated by E237 and may numbers of T cells in the vagino-cervix mucosae of women does be associated with reduced cell-mediated immune responses. not change during the various phases of the menstrual cycle. Thus it seems that increased levels of P4 during the menstrual Cytotoxic T-cell activity in the cervix and vagina of women is cycle (and progestins delivered via hormonal contraceptives) demonstrated during all phases of the menstrual cycle. may have profound effects on the outcomes of bacterial Thus, both the human LGT and UGT are immunologically infections of the FGT, including those caused by Chlamydia,by active throughout the normal menstrual cycles of women. increasing Treg freqencies and lowering cell-mediated immu- Localization of T-lymphocyte populations in the human female nity responses in these infected populations.123 Infections of the UGT and LGT and the effects of menstrual cycle phases and of human FGT with C. trachomatis are, however, less likely to chlamydial infections on these immune cells is summarized occur in P4-dominant conditions. This is due to the fact that, as (Table 1). previously mentioned, the vaginal epithelia of humans is not as permeable to antigen uptake during the P4-associated luteal HORMONAL REGULATION OF IMMUNOGLOBULIN phase of the menstrual cycle, as P4 does not significantly thin SECRETION this mucosal epithelium. In humans, the predominant antibody isotype in female Decreases in the numbers of Tregs have been reported in cervicovaginal secretions is IgG,129 although secretory IgA in endometrial samples of 15 progestin-treated patients with the polymeric form is also present.130 Levels of IgG and IgA atypical endometrial hyperplasia. In these same patients, antibodies in FGT fluids are modulated by the stage of the progestin treatment was associated with significant increases menstrual cycle in women and the estrous cycle in rodents. in cytotoxic T-cell activity, suggesting that sub-populations of It is known that the neonatal Fc receptor, FcRn, in the genital lymphocytes can be affected by progestins.124 P4 and, to a lesser epithelium mediates transport of IgG into the FGT and that extent, E2 have both also been reported to reduce Foxp3 þ Treg local levels are affected by stage of the murine estrous cycle, with cells in peripheral blood mononuclear cells in in vitro systems highest levels being recorded at diestrous.131 The transport of that represented the second trimester of human .125 IgG into the fallopian tubes and uterus is also significantly T-cell activation signalling (by CD3zeta, Janus-associated decreased by P4 treatment, and this is likely correlated with kinase 2 (JAK2), and JAK3 proteins) and cytokine production FcRn expression.132 in cells exposed to hypoestrogenic (4 pg ml À 1 of E2) Mucosa-associated epithelial chemokine CCL28 can recruit postmenopausal levels of E2 were shown to diminish IgA plasma cells to the uterus of the murine reproductive tract significantly, and this correlated with reduced IL-2 production following mucosal (intranasal or vaginal) immunizations, and in Jurkat 6.1T cells.126 This latter finding highlights the fact that this migration is dependent upon E2.133 Interestingly, the IgA a reduction in levels of E2 after menopause can and does plasma cells induced by mucosal vaccination were recruited to contribute to immunosenescence. the uterus but not to the cervix or the vaginal canal. E2 also T-lymphocyte activity in the human uterus is also influenced upregulates the in vitro expression in the FGT of secretory by the menstrual cycle; e.g., CD3 þ CD8 þ CTL activity is component increasing transport of IgA into the lumen,134 and detectable during the proliferative phase but absent during the transport of polymeric IgA (pIgA) into FGT tissues is secretory (postovulatory) phases of fertile women.33 In significantly decreased by ovariectomy, although this decline postmenopausal women, strong CD3 þ T-cell cytolytic activity can be reversed by the addition of E2 but not of P4.132 is detected in all compartments of the FGT. These findings The levels of Igs in genital tract secretions of women vary suggest that the high levels of E2 and P4 present during the during the menstrual cycle, with the lowest levels of IgG proliferative phase of the menstrual cycle can downregulate occurring around the time of ovulation in women with human CTL activity in the uterus. Studies show that E2 greatly papillomavirus (HPV)-16 vaccine–specific antibodies with increases the activity of the IFN-g promoter in human T-cell total IgG and IgA titers in cervical fluid highest in the lines, suggesting that E2 may favour Th1 responses. Receptors proliferative phase of the cycle.135,136 In normal cycling female for E2 have also been shown on both CD8 and CD4 T-cell macaques, it has been reported that the frequency of populations.125 Ig-secreting cells (ISC) was significantly higher in tissues Hormonal fluctuations during the estrous cycle that affect collected from animals in the peri-ovulatory period of the cell-mediated immunity in the upper FGT may also potentially menstrual cycle than in tissues collected from other stages of the

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cycle and that this effect was apparently due to the action of There are numerous reports in the literature on studies in estrogen on CD8 þ T cells.137 The effects of ovarian hormones women and in animal models that report the effects of sex on (ISC) function in healthy women were also investigated, and hormones on genital tract infections caused by these mucosal it was reported that ISC frequency in PMBC was highest during pathogens (e.g., for HIV review, see Hel et al.148). the peri-ovulatory stage of the menstrual cycle.138 In this review, we will specifically focus our discussions on In murine studies, not surprisingly, it has been shown that E2 C. trachomatis as this organism is the perennial target for much influences Ig levels in post-pubertal female C57BL/6 (B6) mice, of our own research. with these animals expressing genes involved in adaptive immune responses, including IgA, IgG1, IgG2, and B-cell C. trachomatis receptor gene involved in B-cell signalling.139 In female BALB/c The columnar ECs of the FGT are target cells for chlamydial mice, E2 strongly stimulated the production of bovine serum infection, and although natural immunity to infection with this albumin (BSA)-specific antibodies (mostly of the IgG1 isotype) mucosal pathogen does occur, it is serovar-specific and in the sera of BSA-immunized mice.140 A study of the effects of short-lived. Local host defence against Chlamydia involves sex hormones on migration of antibody-secreting cells into the the mucosal barrier comprised of ECs and and innate, genital tracts of female BALB/c mice revealed that the mRNA humoral, and cell-mediated immune responses (Figure 3). expression from adhesion molecules and chemokines Decreased levels of SLPI in vaginal secretions are reportedly intercellular adhesion molecule 1, P-selectin, and JAM-1 inversely related to infections caused by C. trachomatis,149 but (junctional adhesion molecule 1) was significantly increased SLPI expression is reportedly induced following C. trachomatis in ovariectomized mice and that these changes were restored to infection of the FGT150 and elafin mRNA expression is normal levels by E2 treatment.141 upregulated in a Chlamydia-infected oviduct EC line62 (see The maturation, selection, and activation of B cells is also Table 3150–153). known to be affected by estrogen treatment, with E2 shown to Infection of the FGT with live replicating C. trachomatis is alter the later stages of B-cell development (reviewed in innately recognized by surface PRRs TLR2 and TLR4 that are Grimaldi et al.142). Recently, it has been reported that in highly expressed in the UGT tissues.45 The myeloid female B-cell-deficient mice that do not express IgM, E2 differentiation primary response gene (MyD)88 is essential increased the frequency of IgA-producing plasma cells, for nuclear factor-kb (NF-kb) signalling and transcription of suggesting that E2 can influence an alternative B-cell pathway proinflammatory cytokines. Along with TLR2, MyD88 found in these mice.143 specifically co-localizes with the intracellular chlamydial IgA-secreting plasma cells typically produce dimeric IgA inclusion.154 Additional to the TLRs the NOD-like receptor, (dIgA), and once secreted into the , dIgA binds NOD1, also senses chlamydial infection of endocervical ECs. the polymeric Ig receptor (pIgR) at the basolateral surface of Signals transduced from these TLRs lead to induction of IFN-g mucosal ECs forming secretory IgA. It is known that dIgA:pIgR and cytokine genes that contribute to resolving chamydila complexes can neutralize intracellular pathogens such as genital infection in an IFN-g-dependent manner, noting also HIV,144 although the precise effects of these complexes on that TLR2 is also responsible for severe tubal immunopathology chlamydial infections is presently unknown. The pIgR is in a murine model and that the chlamydial plasmid has a crucial ubiquitously expressed by ECs in the human FGT, with high role in TLR2 activation.155,156 In response to chlamydial expression observed in the UGT.145 Innate (mediated by TLR3) infection, ECs secrete pro-inflammatory cytokines/chemokines and adaptive immune responses (including IFN-g) to infection such as IL-6 and IFN-g that are hypothesized activators are known to regulate pIgR expression. Additionally, the sex of pathology in the cellular paradigm of chlamydial hormones E2 and P4 also have regulatory roles in the pathogenesis.157 expression of pIgR. In female rats, E2 is known to upregulate C. trachomatis infection of the female LGT stimulates innate the pIgR expression,146 although in primates ovulation immune cells by activation of TLR2/TLR4 (reviewed in Schaefer coincides with a decrease in mucosal antibodies.134 The sex et al.158). TLRs 2 and 4 have been found at the highest expression hormone P4 downregulates pIgR expression in rats,147 and levels in endometrium and tissue,159 with the although the specific role of the pIgR in chlamydial infections is predominant expression of TLR4 and its co-receptor CD14 in debatable, the protective role of IgA and the pIgR may indeed be the fallopian tubes proposed to have an important role in the of importance in the development of subunit vaccines against innate host defence mechanism against ascending C. tracho- C. trachomatis. matis infections.158,160 In a recent study, C. trachomatis– infected women have been reported to express higher TLR2, SEX HORMONES AND GENITAL TRACT INFECTION TLR4, and iNOS in their cervical monocytes compared with It is becoming increasingly well documented that sex hormones controls. It is thought that TLR4 initiates the innate immune E2 and P4 regulate host susceptibility, as well as innate and response to chlamydial infection, activation of TLR2 leads to adaptive immune responses, to sexually transmitted pathogens expression of inflammatory cytokines while iNOS contributes to infecting the mucosal surfaces of the FGT. STD pathogens that chlamydial clearance from this mucosal site.161 infect this site include viruses (HIV-1, HSV-2, HPV) and Studies into C. trachomatis infections of the FGT have bacteria such as Neisseria gonorrhoeae and C. trachomatis. reported many effects of hormones on infection and immunity

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Figure 3 Immune protection against chlamydial infection in the female genital tract. Innate, humoral, and cell-mediated immunity act in concert to protect against C. trachomatis infection of non-immune host genital epithelial cells and local innate immune cells in the female genital tract. The hormones estradiol and progesterone regulate innate and adaptive immunity against genital Chlamydia by promoting mucus secretions, regulating immune and non-immune cell numbers and activities (Figure 2), and by altering cell receptor expression at this mucosal site. Innate: The epithelial barrier is relatively ineffective at protecting against Chlamydia as this mucosal pathogen has a myriad of mechanisms to evade barrier protection. A mucus layer containing a variety of antimicrobial factors and endogenous microbiota contributes towards regulating the pH of the FGT to protect against genital tract pathogens. Innate immune cells constitutively secrete an array of soluble antimicrobials, including secretory leukocyte protease inhibitor (SLPI), human b-defensin 2 (HBD2), lysozyme, lactoferrin, Elafin, cathelicidins. Chlamydial infection of columnar epithelial cells and local genital tract immune cells, including neutrophils, macrophages, and natural killer (NK) cells, produces soluble antimicrobials,chemokines, and pro-inflammatory cytokines that selectively prevent bacterial infection of target host cells. (e.g., interleukin (IL)-1 released from infected epithelial cells promotes T helper type 17 (Th17) differentiation). Recruitment and activation of adaptive immune cells (B and T cells) are also orchestrated by the release of these secreted soluble antimicrobial factors from epithelial cells, dendritic cells, and macrophages. Humoral: Antibodies potentially can prevent infection by Chlamydia. Immunoglobulin G (IgG) is the predominant antibody in the FGT. Antibodies released from plasma cells (P IgG and P IgA) inactivate extracellular chlamydial elementary bodies. Cell-mediated: CD4 þ T-cell production of interferon (IFN)-g contributes to host defence by inhibiting intracellular chlamydial replication. Cell-mediated immunity induces apoptosis of infected cells by cytotoxic CD8 þ T cells.6,20,35,39,40,62,63,77,117–119,150–157,172 CTL, cytotoxic T lymphocyte; GM-CSF, granulocyte-macrophages colony-stimulating factor; MMP, matrix metalloproteinase; GRO-a, growth related oncogene-a; TNF, tumor necrosis factor; Treg, regulatory T cell.

to the pathogen at this mucosal site. For example, significant Hormones can also affect the immune responses to increases in the sensitivities of cervical ECs to infection with chlamydial infections of the FGT in mice. A recent seminal C. trachomatis occur in the later stages of the female menstrual study has reported that IFN-e À / À mice showed significantly cycle.162 It has been reported that chlamydial infection increased levels of chlamydial growth in the UGTs at 30 days occurring in the early E2-dominant phases of the cycle was post intravaginal infection with C. muridarum. The mice also a significant predictor for development of salpingitis.163 showed more severe clinical signs of disease following Clinical findings have also reported on the enhancing effects intravaginal infections with sub-lethal doses of C. muridarum. of E2 on chlamydial infection and disease sequelae in the IFN-e expression is also hormonally regulated. The presence of infected FGT. It has been reported that women are more high levels of E2 (proliferative phase) coincided with the susceptible to chlamydial infection under the influence of E2.35 highest expression levels of IFN-e, and E2 administration to In women with fertility disorders (n ¼ 115) and in women with ovariectomized mice induced a six-fold increase in IFN-e C. trachomatis mucopurulent cervicitis (n ¼ 86), a significantly expression. Thus, IFN-e constitutively expressed by luminal positive correlation was recorded between chlamydial load and and glandular ECs of the endometrium can protect against E2 levels.21 Chlamydia without aggravating disease—the first report that

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Table 3 Chlamydia trachomatis and antimicrobial peptides mice, significantly more chlamydiae were recovered from the (AMPs) and oviducts when animals were infected during periods of increased levels of P4 and E2 (secretory phase) rather Study Experimental findings than when infections occurred during the follicular phase.166 Chlamydia stimulates AMP expression Rank et al.127 reported that high E2 levels in guinea pigs King et al.62 In vitro—infection of oviductal epithelial cell line results in corresponded with a significantly greater percentage of increased elafin expression but no effect on SLPI Chlamydia-infected animals developing oviduct pathology. Wheelhouse In vitro—infection of HeLa (cervical epithelium) and JEG- It was also reported that high doses of P4 did not alter the course 150 et al. 3 (trophoblast) cells results in increased SLPI expression of genital infection in guinea pigs,128 but other reports using different animal models showed that these doses increased the 167 AMP activity against Chlamydia rate of infection, e.g., using a rat model and also that high Pal et al.151 In vivo—MMP-7 knockout mice (with reduced doses of P4 resulted in more intense C.muridarum infections of a-defensin activity) were unaffected in their ability to clear mice.168 chlamydial infection Attachment and infectivity of chlamydial elementary bodies Oberley In vitro—surfactant protein D protein inhibits the infection in vitro reportedly has been enhanced by E2 for HeLa cells,169 152 et al. of HeLa cells (an endocervical epithelial cell line by C. human endometrial ECs,170 luminal ECs from swine genital trachomatis 171 153 tracts, and oestrogen-responsive endometrial carcinoma cell Yasin et al. In vitro—rabbit defensin (NP-1), HNP-2, and porcine lines Ishikawa and HEC1B and breast cancer lines.172 In vivo, leukocyte Protegrin reduced chlamydial infection HNP-2, human neutrophil peptide 2; JEG-3, human placental choriocarcinoma cell ECs have been reported to be more sensitive to C. trachomatis line; MMP-7, matrix metalloproteinase 7; NP-1, neutrophil peptide 1; SLPI, secretory serovar E in the E2-dominant (proliferative) stage of the human leukocyte protease inhibitor. menstrual cycle than at other stages of the cycle.173,174 More recently, preliminary work from our own laboratory has indicates a Type 1 interferon can protect and not worsen a investigated infection of ECC-1 human endometrial cell lines chlamydial genital tract disease.69 with C. trachomatis serovars D and L2 supplemented with Chemokine receptors direct T lymphocytes to the site of 200 pg ml À 1 E2 or 20 ng ml À 1 P4. We report that P4 mucosal infection, including infection of genital tract ECs with upregulates adhesion molecules involved in cell recruitment C. trachomatis, and these receptors can be modulated by E2. In (CX3CL1, E-selectin, ITGB8, and others) and downregulates a study examining samples from human patients, it was IFN and IFN receptor genes. Our results indicate that the gene reported that CCR5 expression increased followed C. trachomatis expression patterns induced by E2 in this system are suggestive genital infections.28 It is also now known that CXCR3 of persistent chlamydial infection.175 (chemokine (C-X-C motif) receptor 3) and CCR5 are the Hormonal contraceptive use in women, particularly depo predominant receptors that are crucial for T-lymphocyte access medroxyprogesterone, has also been reported in observational to the genital tract during a C. trachomatis infection in a studies to be associated with a fourfold increase in chlamydial murine model.164 Therefore, the protective capacity of genital tract infections,176 although the biological explanation Chlamydia-specific T cells in the genital tract can be modulated for increased risk of infections caused by depo medroxypro- by the sex steroid E2. gesterone has been challenged.177 A feasibility plan recently has Reproductive hormones may potentially act with cytokines been conducted to pave the way for a future randomized trial to to regulate immune responses in the FGT to chlamydial genital investigate hormonal contraception and the risks of STI tract infections in vivo. Significant negative correlation has been acquisition in women.178 reported between E2 levels in women with primary chlamydial Thus in terms of developing a protective mucosal vaccine for genital infections and cervical wash concentrations of IL-10, Chlamydia, it is, therefore, prudent to consider the influences of IL-1b and IL-6 cytokines. Significant negative correlation was sex hormones on infection and immune responses to also recorded between IL-1b cytokines and P4 levels in women C. trachomatis within the FGTs both of humans and of animals. with recurrent chlamydial infections.165 Thus it would appear that chlamydial infection of the human FGT is modulated, in SEX HORMONE INFLUENCE ON VACCINE-INDUCED part, by the combined actions of cytokines with E2 and P4. IMMUNITY More recent investigations reveal that levels of E2 were Many studies examining the effects of sex hormones on significantly higher in Chlamydia-positive women with fertility vaccine-induced immunity against sexually transmitted patho- disorders when compared with fertile women, suggesting that gens have been conducted using the murine model with this sex hormone contributes to the development of disease animals that undergo an estrus cycle. The cycle consists of sequelae to C. trachomatis infection of the FGT.35 Proestrus and Estrus, in which follicles grow and mature, the Results of studies into the course of genital chlamydial endometrium develops, and an estrogen surge occurs in infections in animal models have also revealed the effects of conjunction with ovulation. In Metestrus, the corpus luteum reproductive hormones on immune responses and disease and high levels of progesterone are seen, and during Diestrus sequelae. Results of these studies have validated much of the the corpus luteum regresses. In estrus, it is also seen that the clinical data for chlamydial genital infections in women. In endometrium is reabsorbed if conception does not occur while

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in the human menstrual cycle the endometrium is shed. It is to this site will require knowledge of the ovarian steroid be noted, however, that with respect to chlamydial infections of status at the time of immunization in attempts to elicit the FGT there are many differences between the murine protective immunity against a live genital tract infection with models using C. muridarum and the pathogenesis of human Chlamydia. C. trachomatis endocervical infections. Two examples of these In non-human primates, studies have shown that live differences are (i) the strain of Chlamydia used in the mouse attenuated lentivirus (simian-human immunodefiency virus) model, which is not a natural pathogen of the murine FGT, and protects 60% macaques against challenge with intravaginal (ii) Th1 immunity to C.muridarum in the mouse model that simian immunodeficiency virus. Depo-provera pre-treatment causes significant damage to the UGTs of mice while in of the animals resulted in a decrease in this protection,184 a endocervical infections of women with C. trachomatis UGT finding that is similar to that observed in mice. damage occurs infrequently. Finally, only a limited number of studies to date have When E2 prevents the penetration of antigen into the vaginal examined the effects of sex hormones on vaccination outcomes epithelium by increasing the mucosal barrier thickness, it in females. Women who were given vaginal immunizations with reduces the antigen-loading of vaginal APCs. E2 thus inhibits bacterial LPS during either the E2-dominant follicular or the P4- the crucial process of CD8 þ T-cell priming, although antigen associated luteal phases of the ovarian cycle only induced cervical uptake can still occur in the vaginal mucosa following removal IgA2-restricted antibodies against LPS when immunized during of the mucus layer at this site. Interestingly, when antigen is the follicular phase of the cycle.185 Women who were given administered orally or intranasally during the E2-dominant intravaginal immunizations with a recombinant Cholera toxin B phases of the estrus cycle, specific antibodies in the FGT are subunit and immunogen showed no differences in their resulting significantly enhanced.179 The ability to prime CD8 þ T cells immune responses to the antigen whether they were using P4- after immunization at the progesterone-treated genital tract is containing intra-uterine devices, on the oral contraceptive pill, or strongly influenced by ovarian steroid hormones.180 Thus, it were using no contraception at all.186 These results indicate that needs to be remembered that sex steroids can modulate the magnitude of the immune responses elicited in the FGT immune responses following intravaginal and intranasal routes could be influenced by the phase of the menstrual cycle for of immunization, routes that are more effective at eliciting intravaginally administered vaccines. genital tract antibody responses than parenteral immunization. Song et al.181 investigated the effects of exogenous E2 SUMMARY administration, following intravaginal inoculation of BALB/c The FGT is a site of abundant immune processes having mice with N. gonorrhoeae, and they found an increase in an extensive lymphoid system capable of mounting a gonococcal-specific serum antibody in these animals. Others protective immune response to intracellular pathogens, such as have found that E2 treatment increased the specific antibody C. trachomatis. The effects of ovarian steroid hormones response to Dinitrophenyl–keyhole limpet hemocyanin on innate immunity, antigen presentation, T-cell-specific (DNP–KLH), a T-cell-dependent antigen, in BALB/c mice. immunity, and Ig production have been examined with some By experimenting with agonists to each of the ERs (ERa and studies that have been highlighted presenting contradictory ERb), they determined that the effects of E2 on antibody findings to other studies discussed. The contrary reports are production are mediated via signalling pathways involving likely due to the variety of cell lines and models used in the both ER receptors.182 It appears that the timing between E2 experimental investigations and also due to varying levels and administration and immunization or challenge doses can affect combinations of hormones tested in these studies. the ability of the antigen/micro-organism to penetrate the This review highlights and updates our current knowledge of mucosal epithelia; choice of mouse strain may also affect these the effects of ovarian steroid hormones on various components processes. of immune responses in the FGT and how these responses can Protection against several STIs, including C. trachomatis, modulate susceptibility of this mucosal site to STIs. Future requires not only the stimulation of IgG and IgA antibodies investigations into vaccines to control infections of the female but also antigen-specific CD4 þ T cells in the genital tract. reproductive tract will need to consider the effects both of E2 However, our knowledge is rudimentary regarding both T-cell and of P4 at this mucosal site. Animal models and in vitro assays responses to the effects of sex steroids following mucosal should continue to be refined in design with respect to sex immunizations to induce these responses in the FGT. hormone status in attempts to more accurately reflect the A recent study investigating live chlamydial genital tract potential efficacy of novel vaccine preparations tested in these infections in the murine model reported that P4 is required models that are destined for use in humans. for an effective, protective T-cell response in the genital tract following intravaginal immunizations but that E2 ACKNOWLEDGEMENTS prevented a response.39 Thus CD4 þ T-cell immunity in Australian National Health and Medical Research Council (NHMRC). the FGT can indeed be induced by local mucosal immuniza- DISCLOSURE tion with appropriate chlamydial immunogens (e.g., major The authors declared no conflict of interest. outer membrane protein) and adjuvants (e.g., CTA-DD) as has recently been reported.183 Optimal protection at & 2013 Society for Mucosal Immunology

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